In Sudanese traditional medicine Acacia seyal is a very important plant, it has valuable uses in cosmetic and for treating of many diseases and infections. The pleasantly fragrant stem fumigate bath, known as Dokhan, is used for cosmetic and medicinal purpose, mainly for the treatment of vaginal and urinary tract infections. Searching for new sources of naturally occurring antioxidants and antimicrobial become of interest, due to the carcinogenic effect of synthetic antioxidants, and resistance of antimicrobial agents by microorganisms. The present study represents the first attempt to investigate the content and chemical composition, antioxidant and antimicrobial activities of the dry distillate of A. seyal stem. The brown dry distillate (4.0 %v/w) was prepared using dry distillation method. The antioxidant activity assayed by using DPPH radical scavenging was found to be 94±0.01% compared to propylgallate, standard antioxidant agent 90±0.01. The results of antimicrobial using disc diffusion techniques showed remarkable activity with inhibition zone range of 15-25mm. The constituents of the oily dry distillate investigated by GC-MS technique showed detection of one hundred and twenty two compounds. The main constituents identified are Solerone (7.27%), Furfural (7.15%), Catechol (7.11%), Syringol (5.56%), Allo-Inositol (4.86%), Mequinol (4.81%), Furfuralcohol (3.35%), 3-Methyl-1,2-cyclopentanedione (3.24%), Phenol (2.73%), Homovanillyl alcohol (2.56%) and 3-Cresol (2.11%).